This invention relates to biosensors that are useful in detecting chemical compounds of interest. Such biosensors are receptors such as G-protein coupled receptors, tyrosine kinase receptors, and/or ion channels, selected via mutagenesis. More specifically, the biosensors of the invention are highly specific and highly sensitive in detecting low levels of the chemical compounds of interest.
Current methods for detecting chemical compounds of interest that offer the greatest sensitivities, such as mass spectrometry and chromatography, require cumbersome fragile equipment that need regular maintenance and calibration. These conventional assays are also limited by the specificity of the methods, the possibility of false positive detection of structurally-related compounds and the speed of chemical detection.
Natural biosensors are intricate biological systems that have evolved over billions of years to discriminate between chemical structures, to sense small numbers of molecules and to register a response in less than a second through amplification of the signal within the cell. These natural biosensors work through protein receptors.
The most common example of such highly-discriminating sensors are the olfactory receptors which are members of the G-protein coupled receptor (GPCR) superfamily (Buck, L. and Axel, R. (1991) Cell 65:175-87). The nose is the most sophisticated chemical sensor ever devised. In less than a second a nose can detect and distinguish between vast numbers of chemicals. Nature""s unrelenting application of the evolutionary paradigmxe2x80x94selective pressure for survival of the fittestxe2x80x94has honed this instrument to perfection. For example, salmon use biosensors to return to their specific birth streams and a male moth, using one to three highly specific pheromone receptors can track and locate a single female several miles away. Other animals have developed the ability to distinguish thousands of distinct molecules using a complement of approximately 1,000 receptors. Dogs are routinely used for detecting explosives, illicit substances and for locating victims buried in the rubble of natural or man-made disasters.
Besides their contribution to olfaction, the importance of GPCRs to higher organisms including humans can be noted in the fact that 2,500 of the roughly 100,000 genes encoded in the human genome are for GPCRs (including the 1,000 for olfaction). An immense range of structurally diverse ligands are detected by the GPCRs. In addition to thousands of odorants comprised of naturally occurring and synthetic chemicals, GRCR ligands include structures from sugars (sucrose) to lipids (prostaglandins, leukotrienes) to peptides (from dipeptidesxe2x80x94Nutrasweet xe2x80x94to proteins of 10 kD) to ions (calcium) to small aromatic molecules (melatonin, catecholamines, etc.) and even photons. This diversity of known ligands suggests that the range of chemical structures that can be detected by suitably-evolved receptors is unrestricted.
Perhaps not surprisingly, when small molecules activate GPCRs, they appear to do so by binding to amino acids located deep in their transmembrane regions. The panoply of GPCRs seen today have broadly similar structural motifs. For example, the seven membrane spanning regions of bacteriorhodopsin define an elliptical pocket (Roper, D., Jacoby, E., et al. (1994) Journal of Receptor Research 14:167-86). It is within this well that the photosensitive retinal (ligand) lies. Retinal is covalently attached to a lysine on transmembrane domain seven. This protein, along with its relatives halorhodopsin and sensory rhodopsin, comprise an ancient class of bacterial proteins that respond to photons by pumping protons and chloride ions, and by activation of a second protein respectively. All modem GPCRs apparently share the overall structure of these photoresponsive molecules including both the seven transmembrane regions and the corresponding intracellular and extracellular domains.
Small ligands, such as the retinal of rhodopsins, bind to GPCRs within wells defined by the GPCR""s seven transmembrane spanning domains. This has been carefully delineated in a few cases such as the one described for the xcex22 adrenergic receptor (Strader, C. D., et al., (1989) Amer. J. Resp. Cell and Molec. Biol. 1: 81-85).
Although the constitution of the seven transmembrane domains of GPCRs is limited by requirement for overall hydrophobicity, the range of amino acid variation within the transmembrane regions, from receptor to receptor, varies greatly. In all cases there is an overall pattern of hydrophobic and hydrophilic amino acids as required by the alpha helical nature of the sequences. For the most part, hydrophobic amino acids are required for the face of each transmembrane domain that faces outward towards the lipid bilayer. The amino acids facing inward show greater variability. Not surprisingly, receptors with the same ligand, such as the xcex21-3 receptors have greater sequence homology to each other than to disparate receptors such as those for olfaction or gastrin releasing peptide (GRP). As with an antibody selected following immunization with a particular compound, there is no clear a priori correlation between the structure of the ligand, in terms its physico-chemical properties, and the general structural features of the receptor.
The xcx9c1,000 olfactory receptors, taken together, recognize over 10,000 different chemicals including many synthetic, non naturally occurring ones such as numerous odorous organic molecules developed by the chemical industry. Different GPCRs such as the dopamine 1 and 2 receptors share the same ligand, yet the two receptors are only somewhat related. Meanwhile, one receptor may be activated by more than one ligand with varying degrees of similarity. Both the number and diversity (or alternatively the degree of focus) of the set of chemicals used to drive the selection of the set of receptors to be used in a sensor influence the range and specificity of the final sensor.
There is a need for highly specific and highly sensitive sensors that detect, a range of chemical compounds.
There is a need for sensors that detect, within a short period of time, a range of chemical compounds.
There is an additional need for standard analytical methods to monitor products for authenticity, or compliance to standards.
The invention provides novel methods for identifying mutated receptors, novel methods for testing a sample for the presence of a ligand, novel methods for generating and identifying a fingerprint for a ligand and novel detectors for identifying the presence of a ligand which binds to a cell surface receptor. The invention also relates to methods for analyzing products based upon the presence of ligands in such products that are constituents of the products. These methods allow for providing a xe2x80x98signaturexe2x80x99 for the product, enabling authentication and monitoring of products for safety, security purposes, fraud and quality control. Other aspects of the invention will be readily apparent to those of ordinary skill in the art from a reading of the detailed description of the invention.
According to one aspect of the invention, a method is provided for identifying a mutated receptor that binds the ligand. First there is obtained or there is generated a plurality of nucleic acids that code for a plurality of mutated receptors. The plurality of nucleic acids then are introduced into a plurality of cells. It is preferred that the cells in their natural state do not generate a signal when contacted with the ligand. There are different nucleic acids in different of the plurality of cells. The plurality of cells then are contacted with the ligand. An intracellular signal in a cell, generated by a ligand binding to one of the plurality of mutated receptors, is detected. The signal is indicative of the presence of a mutated receptor that binds the ligand, when the mutated receptor is selected from the group consisting of mutated G protein coupled receptors, tyrosine kinase receptors, and ion channels.
In one embodiment, the signal detected can be compared to a signal generated by a cell expressing a non-mutated receptor of the same type as the mutated receptor, thereby permitting the identification of a mutated receptor with an altered binding specificity for the ligand versus the non-mutated receptor. In another embodiment, the signal can be compared to a cell expressing a different mutated receptor which binds the ligand. In one embodiment, the ligand is not the natural ligand for the receptor.
The signals generated can be second messenger signals, which are well known to those of ordinary skill in the art. Such signals include those that result in pigment dispersion and those that cause alterations in calcium levels in the cell. Thus, the signal detected in some embodiments can be pigment dispersion and/or aggregation or calcium mediated fluorescence. Such assays are well known to those of ordinary skill in the art. Where the signal is pigment dispersion and/or aggregation, the cells preferably are melaniferous and most preferably are lower animal pigment cells. Where the signal is calcium mediated fluorescence, the cells can be virtually any cell known to those of ordinary skill in the art which have altered calcium levels as a result of the foregoing receptors. Fibroblasts, 3T3 cells, lymphocytes, keratinocytes, etc., may be used. The mutated receptors also can be cloned into yeast cells, and assays involving the propagation of the yeast known to those of ordinary skill in the art can be employed as the detectable signal. Likewise, RSAT systems such as those described in U.S. Pat. No. 5,707,798, entitled xe2x80x9cIdentification of ligands by selective amplification of cells transfected with receptors,xe2x80x9d issued Jan. 13, 1998, to Brann, M R, can also be employed.
According to another aspect of the invention, a method is provided for testing a sample for the presence of a ligand. The method involves contacting the sample with an exogenous cell surface receptor mutated to have altered binding to its natural ligand, and determining the presence of a preselected signal generated if the ligand binds to the exogenous cells surface receptor. Preferably, the mutated receptor is part of a recombinant cell expressing the receptor. In one important embodiment, the recombinant cell is at least two recombinant cells, each expressing a respective exogenous cell surface receptor mutated differently to have differently altered binding to the natural ligand. In another important embodiment, the recombinant cell is an array of recombinant cells expressing an array of differently mutated exogenous cell surface receptors.
Important receptors, cell types, signals, and so on are as described above.
According to still another aspect of the invention, a method is provided for generating and identifying fingerprint for a ligand. The method involves generating a plurality of signals by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors, each of said receptors having a different selectivity or specificity for the ligand, the plurality of signals comprising the fingerprint. The fingerprint can take on any of a variety of forms. The fingerprint may be a fluorescence read-out, may be a spatial pattern, may be a graph, and so on. It is important only that the combination of the signals be derived from binding of a ligand to the array, any particular ligand generating a different pattern when contacted with the array of recombinant cells.
According to yet another aspect of the invention, a detector for identifying the presence of a ligand which binds to a cell surface receptor is provided. The detector includes a container housing a cell culture medium. Cells are contained in the container, the cells expressing a receptor which binds the ligand and producing a detectable intracellular signal when the ligand binds the receptor. The container also has an inlet port for introducing a sample containing the ligand into the container. A sensor is attached to the container for detecting the intracellular signals. In one important embodiment, the receptor is an exogenous cell surface receptor. In another important embodiment, the receptor is an exogenous cell surface receptor mutated to have altered binding to its natural ligand. In a particularly important embodiment, the cells are an array of cells expressing an array of mutated receptors.
According to a further aspect of the invention, a method is provided for determining relatedness of a sample to a standard known to be authentic or known to have at least one selected characteristic of authentic material. The method involves generating a plurality of signals for a ligand-containing standard by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors. Each of the receptors has a different selectivity or specificity for a ligand in the ligand-containing standard. The plurality of signals comprises a standard-fingerprint for the ligand-containing standard.
The method further involves generating a plurality of signals for a ligand-containing sample by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors. Each of the receptors has a different selectivity or specificity for a ligand in the ligand-containing sample. The plurality of signals comprises a sample-fingerprint for the ligand-containing sample. It is also a requirement that the array of recombinant cells expressing an array of exogenous mutated cell surface receptors contacted by the ligand-containing sample is identical to the array of recombinant cells expressing an array of exogenous mutated cell surface receptors contacted by the ligand-containing standard.
The method finally involves, comparing the sample-fingerprint with the standard-fingerprint to determine whether the ligand-containing sample is authentic.
In some embodiments, the chemical composition of the ligand-containing standard is unknown. In certain embodiments, the ligand-containing standard comprises a plurality of ligands, each ligand binding to a different array of recombinant cells expressing an array of exogenous mutated cell surface receptors. In preferred embodiments, a pattern of signals from the sample-fingerprint is compared to a pattern of signals from the standard-fingerprint, and authenticity requires the pattern of signals from the sample-fingerprint to be within a pre-selected confidence limit defining a range of a pattern of signals calculated from the pattern of signals from the standard-fingerprint. In further embodiments, the comparing step is carried out by a microprocessor. In yet further embodiments, each of the fingerprints can be a fluorescence read-out, a spatial pattern, or a graph.
According to another aspect of the invention, a computer-implemented method for determining identity of a product, is provided. The method involves receiving standard-fingerprint data produced by generating a plurality of signals for a ligand-containing standard by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors, each of the receptors having a different selectivity or specificity for a ligand in the ligand-containing standard. The plurality of signals comprises a standard-fingerprint for an authentic ligand-containing standard.
The method further involves, receiving sample-fingerprint data produced by generating a plurality of signals for a ligand-containing sample by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors, each of the receptors having a different selectivity or specificity for a ligand in the ligand-containing sample. The plurality of signals comprises a sample-fingerprint for the ligand-containing sample. It is also a requirement that the sample-fingerprint data is generated using the same array of recombinant cells expressing an array of exogenous mutated cell surface receptors contacted by the ligand-containing standard to generate the standard-fingerprint data.
The method finally involves, comparing sample-fingerprint data from the ligand-containing sample to standard-fingerprint data from the ligand-containing standard, and identity so requires the sample-fingerprint data from the ligand-containing sample to be within a pre-selected confidence limit defining a range of values calculated from the standard-fingerprint data.
In important embodiments, the computer-implemented process further comprises using a computer database for storing and making available information about standard-fingerprint data of an authentic product and includes a computer-readable medium having computer-readable logic stored thereon. The computer-readable logic comprises a plurality of records for the authentic product indicating measurements of a plurality of signals for a ligand-containing standard generated by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors. Each of the receptors has a different selectivity or specificity for a ligand in the ligand-containing standard. The plurality of signals comprises a standard-fingerprint for an authentic ligand-containing standard, and an indication of the product. The records are accessible using an indication of the product, wherein the step of receiving standard-fingerprint data for the authentic product includes the step of accessing the computer-readable medium using an indication of the product to retrieve the records.
According to yet another aspect of the invention, a computer database for storing and making available information about standard-fingerprint data of an authentic product, is provided. The computer database comprises a computer-readable medium having computer-readable logic stored thereon. The computer-readable logic comprises a plurality of records for the authentic product indicating measurements of a plurality of signals for a ligand-containing standard generated by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors, each of the receptors having a different selectivity or specificity for a ligand in the ligand-containing standard, the plurality of signals comprising a standard-fingerprint for an authentic ligand-containing standard, and an indication of the product. Means for accessing the computer-readable medium using an indication of the product to retrieve the records, are also provided.
In the foregoing discussion, the receptors, the assays and the detectors are described in connection with recombinant cells. It should be understood that this represents only a preferred embodiment, and the recombinant receptors can be otherwise provided in arrays on substrates such as microchips. The receptors may be coupled to sensing material such electrotransmissive or conductive polymers and the like. The binding of the ligand then could be detected by a secondary change in the conductive material, triggered by the binding. The ligand also simply could be labeled, and the pattern of binding detected by detecting the label. Those of ordinary skill in the art will readily recognize how arrays of receptors made according to the invention can be assembled onto substrates and used to achieve the benefits of the invention.
In any of the foregoing embodiments, the ligand can include, but is not limited to, chemical warfare agents, explosives, drugs, fragrances, impurities, environmental toxins, and/or pollutants.
These and other aspects of the invention are described in greater detail below.